Fluorescence imaging of biological cell and tissue samples is used to visualize the presence and expression levels of specific antigens, using probes that conjugate antibodies to fluorescent dyes. It is possible to visualize multiple proteins in a given tissue section using probes that target specific antigens of interest, together with one or more histological dyes such as DAPI, a nuclear counterstain. Other targets such as RNA or DNA can be visualized using fluorescent in situ hybridization and oligo-labeled fluorescent probes, respectively.
Fluorescence imaging of a dye involves exciting it with light of a first wavelength band or range of wavelengths, and observing light that it emits in response to this, in a second wavelength band or range of wavelengths. The propensity of a fluorescent dye to emit light in response to excitation at a given wavelength is termed its excitation spectrum. The wavelength distribution of the fluorescent light a dye emits is termed its emission spectrum.
When multiple dyes are used, they are typically chosen to have different excitation spectra, emission spectra, or both, so that by careful choice of the excitation wavelengths and emission wavelengths used, the dye that is being observed can be distinguished. When the spectra of the various fluorescent dyes are not distinct, but overlap substantially in terms of their excitation spectra and emission spectra, it becomes more difficult to determine what dye is associated with the observed emitted light that one observes.
Many samples exhibit endogenous fluorescence emission. That means that when optically excited, the sample itself emits fluorescent light, in addition to the fluorescent light emitted by fluorescent dyes used in connection with antibody-conjugated probes or as a counterstain. This can add further complexity to the above-mentioned determination.
Multispectral imaging of fluorescent samples involves acquiring a series of images of the sample at different excitation wavelengths, emission wavelengths, or combinations of the two. The various images are assembled into an image cube, where two dimensions of the cube correspond to spatial position in the sample, and the third dimension corresponds to the spectrum associated with the various excitation and/or emission wavelengths.